This application is the national phase under 35 U.S.C. xc2xa7371 of PCT International Application No. PCT/FI98/00576 which has an International filing date of Jul. 8, 1998 which designated the United States of America.
This invention relates to recombinant-DNA-technology. Specifically this invention relates to new recombinant yeast cells transformed with SEB1 gene or its homologs. A yeast cell transformed with several copies of a SEB1 gene or a gene homologous to SEB1 has an increased capacity to produce secreted foreign or endogenous proteins.
Further, said new recombinant yeast cells, when transformed with genes expressing suitable hydrolytic enzymes can hydrolyze and/or utilize appropriate macromolecular/polymeric compounds more efficiently, which results in increased cell mass production and/or more versatile utilization of the compounds in relevant biotechnical applications.
The development of recombinant DNA methods has made it possible to produce proteins in heterologous host systems. This possibility greatly facilitates production of e.g. proteins of therapeutic importance which normally occur in nature in very low amounts or are otherwise difficult to isolate or purify. Such proteins include growth factors, hormones and other biologically active proteins or peptides which traditionally have been isolated from human or animal tissues or body fluids e.g. blood serum or urine. The increasing danger of the presence of human pathogenic viruses such as HBV, HIV, and oncogenic viruses, prions, or other pathogens in the human or animal tissues or body fluids has greatly speeded up the search for heterologous production systems for these therapeutics. Other proteins of clinical importance are viral or other microbial or human parasite proteins needed for diagnostics and for vaccines especially of such organisms which are difficult to grow in vitro or in tissue culture, or are dangerous human pathogens. These include viruses like HBV, HIV, yellow fever, rubella, FMDV, rabies, and human parasites such as Plasmodium falciparum causing malaria.
A further group of proteins for which heterologous production systems have been or are being developed are secreted enzymes, especially those hydrolyzing plant material, and which are needed in food and fodder production as well as in other industrial processes including textile industry and pulp and paper industry. The possibility of producing proteins in heterologous systems or production of endogenous proteins in genetically engineered cells increases their yields and greatly facilitates their purification and has already by now had a great impact on studies of structure and function of many important enzymes and other proteins. The production and secretion of foreign hydrolytic enzymes in yeast for example, results in improvements in processes based on industrial yeast strains such as distiller""s, brewer""s or baker""s yeasts.
Various production systems have been and are being developed including bacteria, yeasts, filamentous fungi, animal and plant cell cultures and even multicellular organisms like transgenic animals and plants. All of these different systems have their advantages, even if disadvantages, and all of them are needed.
The yeast Saccharomyces cerevisiae is at the moment the best known eukaryote at genetic level. Its whole genome sequence became public in data bases on Apr. 24, 1996. As a eukaryotic microbe it possesses the advantages of a eukaryotic cell like most if not all of the post-translational modifications of eukaryotes, and as a microbe it shares the easy handling and cultivation properties of bacteria. The large scale fermentation systems are well developed for S. cerevisiae which has a long history as a work horse of biotechnology including production of food ingredients and beverages such as beer and wine.
The yeast genetic methods are by far the best developed among eukaryotes based on the vast knowledge obtained by classical genetics. This made it easy to adopt and further develop for yeast the gene technology procedures first described for Escherichia coli. Along other lines the methods for constructing yeast strains producing foreign proteins have been developed to a great extent (Romanos et. al., 1992).
Secretion of the proteins into the culture medium involves transfer of the proteins through the various membrane enclosed compartments constituting the secretory pathway. First the proteins are translocated into the lumen of the endoplasmic reticulum, ER. From there on the proteins are transported in membrane vesicles to the Golgi complex and from Golgi to plasma membrane. The secretory process involves several steps in which vesicles containing the secreted proteins are pinched off from the donor membrane, targetted to and fused with the acceptor membrane. At each of these steps function of several different proteins are needed.
The yeast secretory pathway and a great number of genes involved in it have been elucidated by isolation of conditional lethal mutants deficient in certain steps of the secretory process (Novick et al., 1980; 1981). Mutation in a protein, needed for a particular transfer step results in accumulation of the secreted proteins in the preceding membrane compartment. Thus proteins can accumulate in the cytoplasm, at ER, Golgi or in vesicles between ER and Golgi, or in vesicles between Golgi and plasma membrane.
More detailed analysis of the genes and proteins involved in the secretory process has become possible upon cloning the genes and characterization of the function of their encoded proteins. A picture is emerging which indicates that in all steps several interacting proteins are functioning. The number of genes is rapidly increasing that are involved in protein secretion and that were first identified in and isolated from S. cerevisiae and were later found in other organisms including lower and higher eukaryotes. The structural and functional homology has been shown for many of such proteins.
We have recently cloned a new yeast gene, SEB1 (Toikkanen et al., 1996) which encodes the xcex2-subunit of the trimeric Sec61 complex (hence the name: SEB=SEc61 Beta subunit) that is likely to represent the protein conducting channel of the ER both in co- and post-translational translocation (Hanein et al., 1996). In the former it functions in close connection with the ribosome and in latter it forms a heptameric membrane protein complex with the tetrameric Sec62/Sec63 complex (Panzner et al., 1995). Genes with sequence similarity to the SEB1 gene are found in plant and mammalian cells indicating that the Sec61 translocation complex is conserved in evolution. In fact, similar components function in protein translocation also in prokaryotes (discussed in Toikkanen et al., 1996). This further supports the conserved and central role of the SEB1 gene in protein secretion and intracellular transport. However, no reports exist so far on any positive effect of the SEB1 or its homologs in other yeasts, plant or animal cells on secretion when overexpressed, which effect we are showing in this invention for the yeast SEB1 gene. It should be noticed that Seb1 protein is present in a different protein complex and at different location than the Sso proteins which we have previously shown to enhance production of secreted proteins when present in the cells in higher than normal amounts.
Knowledge on the protein secretion process in S. cerevisiae is rapidly increasing. Less is known about the secretory system of other yeasts such as Kluyveromyces, Schizosaccharomyces, Pichia and Hansenula which, however, have proven useful hosts for production of foreign proteins (Buckholz and Gleeson, 1991; Romanos et al., 1992), or Candida and Yarrowia which also are interesting as host systems. The genetics and molecular biology of these yeasts are not as developed as for Saccharomyces but the advantages of these yeasts as production hosts are the same as for Saccharomyces.
Several attempts have been made and published previously to increase foreign protein production in yeast and filamentous fungi as well as in other organisms. Much work has been devoted to various promoter and plasmid constructions to increase the transcription level or plasmid copy number (see e.g. Baldari et al. 1987; Martegani et al. 1992; Irani and Kilgore, 1988). A common approach to try and increase secretion is to use yeast signal sequences (Baldari, et al. 1987, Vanoni et al. 1989). Random mutagenesis and screening for a secreted protein (Smith et al., 1985; Sakai et al., 1988; Shuster et al., 1989; Suzuki et al., 1989; Sleep et al., 1991; Lamsa and Bloebaum, 1990; Dunn-Coleman et al., 1991) or fusion of the foreign protein to an efficiently secreted endogenous protein (Ward et al., 1990; Harkki et al., 1989; Nyyssxc3x6nen et al. 1993; Nyyssxc3x6nen et al., 1992) have been widely used both for yeast and filamentous fungi in order to make the secretion of foreign proteins more efficient. Both of these methods are of limited use. Overproduction mutants isolated by random mutagenesis and screening are almost exclusively recessive and thus cannot be transferred into industrial yeast strains which are polyploid. Often the overproduction results from changes other than increased secretion and in many cases affects only the protein used for screening. Fusion protein approach requires tailoring of the fusion construction for each foreign protein separately. The fusion protein is often not functional and thus the final product must be released by proteolytic cleavage which complicates the production procedure.
Our approach, increasing the copy number of genes functioning in secretion and thus the amount of components of the secretory machinery is more universal: it is applicable to any protein without specific fusion constructions and applicable to diploid and polyploid strains.
It is not exactly known which steps form the bottle necks in the secretory process, but it can be anticipated that there are more than one stage that may become rate limiting especially under overproduction conditions. The SEB1 gene according to the invention was cloned using a yeast genetic approach and it was shown to interact genetically with the SEC61 gene, encoding the major component of the ER translocation complex. The fact that overexpression of SEB1 gene increases the production of secreted proteins into the culture medium suggests that the Seb1 protein is a rate limiting component in the translocation process. This was surprising since Seb1 protein is a component of a multiprotein complex and the enhancing effect did not require increased levels of the other components of the complex and since the SEB1 gene is not essential for yeast growth. This could mean that there is another gene which can perform the same function as SEB1. We have isolated another gene, SEB2, homologous to SEB1 but disruption of both SEB1 and SEB2 was not lethal either, indicating that the function of SEB1 is not essential for yeast growth.
The present invention describes a method for enhanced production of secreted proteins based on overexpression of the previously isolated SEB1 gene of Saccharomyces cerevisiae. Specifically, the present invention describes the construction of S. cerevisiae strains overexpressing the SEB1 gene either on a multicopy plasmid or when integrated into the yeast genome in single or multiple copies or placed under regulation of a strong promoter. In addition, this invention describes identification of SEB1 homologs from other yeasts, and detection of Seb1p homologous protein in Kluyveromyces lactis. 
This invention thus provides new recombinant yeast cells expressing enhanced levels of Seb1 protein of S. cerevisiae. 
This invention also provides process(es) for production of increased amounts of secreted proteins by overexpressing genes interacting with the SEB1 gene, such as SEC61.
The yeast cells according to the invention being transformed with the SEB1 gene or genes interacting with the SEB1 gene have an increased capacity to produce secreted proteins. The new yeast cells according to the invention can also be used for more efficient production of hydrolytic enzymes and hydrolysis of e.g. polymeric substrates which results in improvements in biotechnical processes such as single cell or baker""s yeast production due to increased cell mass or in other processes where efficient production of hydrolytic enzymes and/or efficient hydrolysis of plant material is beneficial.